以Tol2系統發展抗菌螢光斑馬魚之研究

Abstract

本研究利用基因轉殖法建立帶有抗菌胜肽epinecidin-1之基因轉殖斑馬魚，藉此探討基因轉殖抗菌胜肽應用於水產養殖的可能。 研究中選用斑馬魚肌凝蛋白輕鏈二 (myosin light polypeptide 2, mylz2) 之啟動子，並以螢火蟲冷光表達系統分析啟動子活性。在2.5、2.3、2.1和1.9 kb之啟動子片段中，發現2.5 kb的活性最高，比已知具最高活性的1.9 kb高約2.8倍。另外也以2.5 kb之啟動子片段成功建立基因轉殖之紅螢光斑馬魚，證明此啟動子片段可大量的表達外源蛋白於骨骼肌之中。 另一方面，進行改良Tol2跳躍基因轉殖法，把有助於轉譯的斑馬魚血球蛋白 (ba1 globin) 之非轉譯區 (untranslated regions, UTRs) 分別構築於Tol2轉位酶 (transposase) 的5' 和3' 端，經瞬時胚期供體跳出分析 (transient embryonic excision assay, TEEA) 與活體螢光表現分析後，證實可提高轉位酶在斑馬魚胚胎發育時期執行轉位之效率。 最後進行抗菌胜肽epinecidin-1之轉殖，以Epi-1/DsRed融合蛋白之形式轉殖入斑馬魚中，並由免疫組織化學染色 (Immunohistochemical stain)，觀察到骨骼肌、血液等組織均有訊號。並在創傷弧菌 (Vibrio vulnificus) 感染後之6、12和24小時進行抗菌分析，結果皆能有效的降低魚體內之菌數。而在細胞激素調控分析實驗中，可以發現Epi-1/DsRed之基因轉殖斑馬魚的內生性MyD88表現量偏高，且在創傷弧菌感染後的12小時，其MyD88、TLR4a之表現量皆高於對照組，反之處於下游基因的IL-1β與TNF-α之表現卻低於對照組，推測Epi-1/DsRed可誘導類鐸受體 (toll-like receptor, TLR) 訊息路徑的負調控。

For the purpose of understanding the application of antimicrobial peptides in aquaculture, transgenic zebrafish expressing epinecidin-1, was developed and reported here. First, we cloned zebrafish mylz2 promoter for this purpose. To characterize the activity of mylz2 promoter, various fragments of the mylz2 promoters were analyzed using firefly luciferase transient expression assay, in which maximum promoter activity was found in the 2.5 kb fragment. Besides, the 2.5 kb fragment also expressed considerable red fluorescent proteins in the skeletal muscle of transgenic zebrafish. Second, in order to improve the translation efficiency of the Tol2 transposase, we constructed the UTRs of zebrafish ba1 globin flanked by the transposase . TEEA and in vivo fluorescent observation, showed high transposition efficiency during embryonic development. After the optimization of promoter and transgenic efficiency, Epi-1/DsRed transgenic zebrafish was developed and expression of Epi-1/DsRed in the muscle and blood were demonstrated by immunohistochemistry staining techniques. Moreover, we also found that Epi-1/DsRed gene was efficiently and significantly expressed in vivo against Vibrio vulnificus 204. Gene expression study in Q-PCR revealed that Epi-1/DsRed itself could induce endogenous MyD88 expression in vivo. After Epi-1/DsRed transgenic zebrafish were infected with Vibrio vulnificus 204, MyD88 and TLR4a were upregulated, but IL-1β and TNF-α were downregulated at 12 hours post infection. It suggest that epinecidin-1 may negatively regulate toll-like receptor signaling pathway.